CN109206491A - Preparation method of porcine pseudorabies virus gD protein, porcine pseudorabies virus subunit vaccine and application - Google Patents

Preparation method of porcine pseudorabies virus gD protein, porcine pseudorabies virus subunit vaccine and application Download PDF

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CN109206491A
CN109206491A CN201710551363.8A CN201710551363A CN109206491A CN 109206491 A CN109206491 A CN 109206491A CN 201710551363 A CN201710551363 A CN 201710551363A CN 109206491 A CN109206491 A CN 109206491A
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porcine pseudorabies
pseudorabies virus
cell
albumen
protein
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钱泓
吴有强
卞广林
张强
徐玉兰
吴素芳
车影
宋月鸿
吕洋萍
闻雪
姜冰洁
查银河
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Novo Biotech Corp
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Abstract

The invention discloses a method for preparing porcine pseudorabies virus gD protein, a porcine pseudorabies virus subunit vaccine and application, wherein the vaccine comprises (1) porcine pseudorabies virus gD protein, and the concentration of the gD protein is 30-200 mug/head part; (2) the concentration of the porcine GM-CSF protein is 10-80 mu g per head part; (3) a pharmaceutically acceptable adjuvant. The method for preparing the gD protein in the vaccine comprises the following steps: 1) cloning the porcine pseudorabies virus gD protein gene subjected to codon optimization into a eukaryotic expression vector to obtain a recombinant plasmid containing the porcine pseudorabies virus gD protein coding gene; 2) then, transfecting recombinant plasmids containing porcine pseudorabies virus gD protein coding genes into CHO cells; 3) obtaining a highly expressed cell line by culturing, screening and acclimating the CHO cell line described in 2); 4) fermenting and culturing the cell strain in the step 3), and purifying to obtain the recombinant porcine pseudorabies virus gD protein. The vaccine has the advantages of industrial production, low cost, easy quality control, good immune effect and the like.

Description

The preparation method and porcine pseudorabies virus subunit vaccine of porcine pseudorabies virus gD albumen And application
Technical field
The present invention is directed not only to a kind of porcine pseudorabies virus subunit vaccine and its preparation method and application, further relates to one kind The Chinese hamster ovary celI strain and its preparation method and application of stability and high efficiency secreting, expressing porcine pseudorabies virus gD albumen, belongs to animal vaccine With veterinary biologics technical field.
Background technique
Porcine pseudorabies are the acute biographies of the pig caused by porcine pseudorabies virus (Pseudorabies virus, PRV) It catches an illness.The disease is in pig break out and spread.It can cause pregnant sow miscarriage, stillborn foetus, the mummification of fetus, boar infertility, newborn piglet Mortality, growing and fattening pigs expiratory dyspnea, growth retardation etc. are one of the serious infectious diseases for endangering global pig breeding industry.
The prevention of the disease mainly passes through vaccine immunity at present, currently on the market popular mainly second generation genetic engineering base Because of deletion of vaccine.The vaccine in non-coding in the gene of glycoprotein in addition to that must introduce other than TK gene introduces a missing One new missing, or one reporter gene of insertion, the mutant strain obtained in this way cannot generate the glycoprotein lacked, because And immune animal cannot generate corresponding antibody, therefore can be wild by immunity inoculation pig and natural infection by serological method The pig of poison distinguishes.This is also the most distinguishing feature of second generation gene-deleted vaccine.It, can be in addition, the missing of some glycoprotein Further decrease virulence.First PRV recombinant vaccine OM-NIVAC-PRV for applying for a patent and registering to use is exactly TK base Because of deletion of vaccine.In China, PRV Ea TK-/gG-, TK-/gE- vaccine strain are constructed on the basis of TK- plants of PRV Ea.But it should The shortcomings that vaccine is also than more prominent: can gene-deleted vaccine cause latent infection or be activated as infectious virus to make us carrying on a shoulder pole Sorrow；Gene-deleted vaccine occurs genetic recombination between wild poison or different gene-deleted vaccines in animal body and is mutated into virulent Strain, to become the new infection sources, this possibility is existing；Gene-deleted vaccine after inoculation there is also latent infection and The problem of toxin expelling.
Pseudorabies virus belongs to herpetoviridae (Herpesviridae), herpesvirus suis category, and virion is circle, 150~180nm of diameter, nucleocapsid diameter are 105~110nm.The outermost layer of virion is virus envelope, it is thin by host The lipid bilayer structure that born of the same parents are derived.Cyst membrane surface has that be about fibre that 8~10nm is arranged radially prominent.Pseudorabies virus base Because group is linear double chain DNA molecule, size about 150kb, average G+C content is up to 74%, with typical hsv gene Group structure feature, by unique long section, unique short section and terminal repeat (TR) and internal repetition sequence positioned at the two sides US Arrange (IR) composition.Have now been found that 11 kinds of PRV glycoprotein, wherein gB, gC, gD are the egg for stimulating body to generate neutralizing antibody It is white, generated antibody either in vivo, in vitro, or in having in the presence of whether there is or not complement and the ability of PRV, Therefore, gB, gC, gD are the preferred glycoprotein for developing PRV subunit vaccine.The advantages of subunit vaccine: nucleic acid substances are not contained Therefore relatively safer；Persistent infection or latent infection will not be generated after inoculation；The immune response of generation can be with wild virus infection phase It distinguishes, is conducive to the control and elimination of epidemic disease.But subunit vaccine also has apparent defect: high production cost, immunogenicity Not as good as attenuated vaccine and inactivated vaccine, using being restricted.
Chinese hamster ovary celI is nineteen fifty-seven Univ Colorado-Boulder USA Theodore doctor T.Puck from an Adult female Hamster ova Separation obtains in nest, is the adherent type cell of epithelium.The cell has immortality, and it is more than generation can to pass on hundred, is current biology Widely used cell in engineering.Relative to other expression systems, Chinese hamster ovary celI has following advantage: (1) having accurately transcription Rhetorical function afterwards, the albumen of expression is in terms of molecular structure, physicochemical property and biological function closest to native protein molecule； (2) not only can adherent growth, but also the culture that can suspend, and have higher tolerance shearing force and osmotic pressure ability；(3) there is recombination base The integration of the efficient amplification and ability to express of cause, foreign protein is stablized；(4) have the function of product exocytosis, and seldom divide The intrinsic protein for secreting itself, isolates and purifies convenient for downstream product；(5) it can be reached with suspension training method or in serum free medium To High Density Cultivation, and volume of culture can reach 1,000L or more, can be mass produced.
Chinese hamster ovary celI type is more, such as: DG44, DXB11, CHO-K1 and CHO-S.Since the 80-90 age in 20th century, Industrially relatively early to use DHFR (dihyrofolate reductase deficiency) gene magnification screening system, host cell strain is DG44.When containing methotrexate (MTX) (methotrexate, MTX) in cell culture medium, dihyrofolate reductase is suppressed, then is led to Feedback regulation is crossed, so that the gene is expanded, and the gene within the scope of its upstream and downstream 100-1,000kb can all expand therewith, Therefore target gene is inserted within the scope of this site can be obtained amplification.The system of many monoclonal antibody productions is still DG44's now DHFR system.GS (glutamine synthelase) amplification system is that one kind for developing in recent years is new using CHO-K1 as host cell Type gene magnification screening system, it has apparent superiority than DHFR system, have been widely recognized in the world at present and It uses.Its principle is GS while ATP is hydrolyzed and provided energy, synthesizes glutamine using intracellular ammonia and glutamic acid.? Lack glutamine culture medium in be added GS inhibitor methionine sulfoxide imonium (L-methioninesulfoximine, MSX), GS gene and the target gene being attached thereto can be made effectively to be expanded, improve destination gene expression level to reach Purpose.The advantages of system, is main: (1) not needing the CHO-K1 cell strain of gene defection type as host cell；(2)CHO- K1 cell is strongr, is easy to cultivate；(3) glutamine decomposition is avoided to cause to cultivate it is not necessary that glutamine is added in the medium The high problem of ammonia level in system, reduces the difficulty of technology controlling and process, and effectively improves cell fermentation density and extend cell life Deposit the time.
When the present inventor uses expressing cho cell gD albumen at the beginning, find the gene of gD albumen without password When son optimization, Chinese hamster ovary celI does not express gD albumen substantially.Therefore, inventors have seen that using expressing cho cell gD When albumen, codon optimization is a necessary process.
Summary of the invention
The technical problem to be solved in the present invention: one be to provide it is a kind of can large-scale industrial production porcine pseudorabies virus The preparation method and application of subunit vaccine；Second is that overcoming existing second generation genetic engineering gene-deleted vaccine in use Existing defect, as gene-deleted vaccine has latent infection and toxin expelling after inoculation；Third is that overcoming existing subunit The generally existing high production cost of vaccine, immunogenicity not as good as attenuated vaccine and inactivated vaccine defect.
According to the first aspect of the invention, the present invention provides a kind of method for preparing porcine pseudorabies virus gD albumen, institutes Preparation method is stated the following steps are included: 1) by the porcine pseudorabies virus gD protein gene cloning after codon optimization to eukaryotic expression In carrier, the recombinant plasmid containing porcine pseudorabies virus gD protein coding gene is obtained；2) porcine pseudorabies virus gD will be contained again The Transfected Recombinant Plasmid of protein coding gene obtains Chinese hamster ovary celI strain into Chinese hamster ovary celI；3) pass through culture, screening, domestication step 2) Chinese hamster ovary celI strain described in obtains the cell strain that height is expressed；4) cell strain highly expressed described in fermented and cultured step 3), Recombinant porcine pseudorabies poison gD albumen is obtained after purification.
In technical solution of the present invention, it is preferable that the porcine pseudorabies virus gD encoding histone base after the codon optimization Because as shown in SEQ ID NO.1.
In technical solution of the present invention, the carrier for expression of eukaryon can for pEE6.4, pEE12.4, pGL4.13, Preferably, the carrier for expression of eukaryon is pEE12.4 to pcDNA3.1.
In technical solution of the present invention, the Chinese hamster ovary celI can be DG44, DXB11, CHO-K1, CHO-S cell strain, excellent Selection of land, the Chinese hamster ovary celI are CHO-K1 cell.
In technical solution of the present invention, it is preferable that in the step 4), highly expressed described in fermented and cultured step 3) The culture medium used when cell strain is the mixed culture medium of CD-CHO and Ex-cell 302, CD-CHO in the mixed culture medium Volume ratio with Ex-cell 302 is 6:4.
According to the second aspect of the invention, the present invention also provides a kind of porcine pseudorabies virus subunit vaccine, the Asia is single Position vaccine includes: (1) porcine pseudorabies virus gD albumen, and the concentration of the gD albumen is g/ parts of 30~200 μ；(2) pig GM-CSF Albumen, the concentration of the GM-CSF albumen are g/ parts of 10~80 μ；(3) pharmaceutically acceptable adjuvant.
In technical solution of the present invention, it is preferable that the concentration of the porcine pseudorabies virus gD albumen be g/ parts of 50 μ~ 100 parts of μ g/.
In technical solution of the present invention, it is preferable that the pig GM-CSF protein concentration is g/ parts of 20 μ~g/ parts of 40 μ.
In technical solution of the present invention, the pharmaceutically acceptable adjuvant can for water adjuvant (such as aluminium glue adjuvant), Oil-in-water adjuvant, water-in-oil adjuvant, W/O/W adjuvant, the preferably described pharmaceutically acceptable adjuvant are ISA201VG。
In technical solution of the present invention, it is preferable that the weight ratio of the ISA 201VG adjuvant and antigen phase is 1:1, described Antigen is mutually the porcine pseudorabies virus gD albumen and the pig GM-CSF albumen.
According to the third aspect of the invention we, the present invention provides a kind of porcine pseudorabies virus gD albumen and porcine pseudorabies again Application of the malicious recombinant subunit vaccine in preparation and dependent diagnostic reagent.
Porcine pseudorabies virus subunit vaccine provided by the invention can induce pig to generate good immune response, and effect is immunized Fruit and existing market seedling are quite even better, which not only has the advantages that such as to be free of possessed by subunit vaccine itself There are nucleic acid substances, therefore relatively safety；Persistent infection or latent infection will not be generated after inoculation；The immune response of generation can be with Wild virus infection is mutually distinguished, and the control and elimination of epidemic disease are conducive to.Also overcome the generally existing immunogene of existing subunit vaccine Defect of the property not as good as attenuated vaccine and inactivated vaccine.In addition also overcoming existing second generation genetic engineering gene-deleted vaccine makes The defect present in process, as gene-deleted vaccine has latent infection and toxin expelling after inoculation.
The present invention constructs and has screened the Chinese hamster ovary celI strain of suspending stabilized efficient secretory expression porcine pseudorabies virus gD albumen, Cell strain expression gD protein yield high (yield is up to 2-3g/L) is easy to purify (as shown in figure 4, in cells and supernatant Destination protein purity is attained by 70% or more, destination protein purity can be made to reach as long as one ni-sepharose purification of simple mistake 90% or more, much meet subunit vaccine preparation the needs of), be easy to be mass produced.Therefore, subunit's epidemic disease is not only solved The problem of another defect, i.e. high production cost existing for seedling, it is sub- to also solve large-scale industrial production porcine pseudorabies virus The problem of subunit vaccine.In addition, this is thin due to using cell strain when producing core component gD albumen in the subunit vaccine Born of the same parents' strain controllability in culture is high, Quality Control is easy, quantitative simple, therefore is also had using the subunit vaccine that this method produces Following advantages: can large-scale industrial production, for amount, sufficient, Quality Control is easy；Stablize between batch；Biosafety control holds in production Easily (without virus, there is no the risks of scattered poison).
Detailed description of the invention
Fig. 1 shows pEE12.4-OPTI-gD plasmid figures；
Fig. 2 indicates pEE12.4-OPTI-gD double digestion qualification result: 1:SpeI/EcoRI double digestion, skeleton size is about 7197bp, clip size about 2632bp, digestion are correct；M:DL10,000；
Fig. 3 indicates the CHO-K1 monoclonal cell strain fermentation supernatant culture medium protein detection of expression PRV-gD albumen: 1 is Marker；2 be the positive control albumen of 1 μ g；3 for 3H8 plant using only Ex-cell302 culture medium fermentation when fermentation supernatant Testing result；4 for 3H8 plant using 60% CD-CHO and 40% 302 mixed culture medium of Ex-cell when fermentation supernatant inspection Survey result；5 for 5G3 plant using only Ex-cell302 culture medium fermentation when fermentation supernatant testing result；6 be 5G3 plants of utilizations Fermentation supernatant testing result when 302 mixed culture medium of Ex-cell of 60% CD-CHO and 40%；7 is individually sharp for 7G11 plants Fermentation supernatant testing result when being fermented with Ex-cell302 culture medium；8 utilize 60% CD-CHO and 40% for 7G11 plant Fermentation supernatant testing result when 302 mixed culture medium of Ex-cell；9 send out for 7D2 plants using only Ex-cell302 culture medium Fermentation supernatant testing result when ferment；10 are mixed for the 7D2 plants of Ex-cell 302 using 60% CD-CHO and 40% Fermentation supernatant testing result when base；11 for 4C2 plant using only Ex-cell302 culture medium ferment when fermentation supernatant detection As a result；12 for 4C2 plant using 60% CD-CHO and 40% 302 mixed culture medium of Ex-cell when fermentation supernatant detection As a result；13 for 6A3 plant using only Ex-cell302 culture medium fermentation when fermentation supernatant testing result；14 be 6A3 plants of utilizations Fermentation supernatant testing result when 302 mixed culture medium of Ex-cell of 60% CD-CHO and 40%；
Fig. 4 indicates protein purification result: M Marker；Supernatant is cell fermentation (culture) supernatant testing result；Flow through for Cell conditioned medium flows through rear testing result；20mM is testing result after the elution of 20mM imidazoles；250mM is to examine after 250mM imidazoles elutes Survey result；500mM is testing result after the elution of 500mM imidazoles；
Pig antibody titer testing result after Fig. 5 indicates immune；
Fig. 6 indicates PRV-gD protein nucleotide sequence and the PRV-gD albumen nucleosides after codon optimization before codon optimization Acid sequence comparison result: OPTI-PRV gD indicates the PRV-gD protein nucleotide sequence after codon optimization；PRV gD indicates close PRV-gD protein nucleotide sequence before numeral optimization.
Specific embodiment
Below with reference to drawings and examples, the present invention will be further described, and the embodiment of the present invention is merely to illustrate this The technical solution of invention, and the non-limiting present invention.
The source list of reagent and drug of the present invention is as follows:
CHO-K1 cell origin is raw in the American Type Culture Collection committee of Chinese Academy of Sciences cell bank Chinese Academy of Sciences Shanghai Order Science Institute's cell bank；
Cell culture medium and serum are purchased from gibco company of the U.S.；
Carrier for expression of eukaryon pEE12.4 is purchased from upper Hailin deep pool Biotechnology Co., Ltd；
Lipofectamine LTX is purchased from U.S. Thermo Fisher company；
Methionine sulfoxide imonium ((L-methioninesulfoximine, MSX)) is purchased from Sigma company；
BCA quantification of protein kit is purchased from U.S. Thermo Fisher company；
ISA 201VG is purchased from match BIC Corp of France.
Embodiment 1: porcine pseudorabies virus gD albumen codon optimization and pEE12.4-OPTI-gD construction of recombinant plasmid
By carrying out codon optimization to porcine pseudorabies virus gD protein nucleotide sequence, OPTI-gD sequence is obtained, such as Shown in SEQ ID NO.1, which is completed.
Sequence after optimization is compared with the sequence (as shown in SEQ ID NO.2) before optimization, discovery there are 214 cores Thuja acid is different, and about 21% (214/1020) nucleotide is different.Specifically as shown in Figure 6.
Embodiment 2:pEE12.4-OPTI-gD construction of recombinant plasmid
2.1PCR expands target fragment OPTI-gD
2.1.1PCR reaction
(1) design of primers and synthesis
Upstream primer: 5 '-CGGAAGCTTATG GCTGACGTGGATGCTGTGCCTG-3 '
Downstream primer: 5 '-GGCGAATTCTTAGTGATGGTGATGGTGATG-3 '
(2) it is loaded 50 μ L of system, as shown in the table:
PCR amplification program:
2.1.2PCR product carries out glue recycling
(1) sample collection EP pipe, adsorption column and collecting pipe have been marked；
(2) the empty EP pipe weight marked is weighed, and records numerical value；
(3) single target DNA band is carefully cut from Ago-Gel with scalpel on bale cutting instrument be put into it is dry In net 1.5mL centrifuge tube；
(4) 600 μ L PC buffer are added in the 1.5mL centrifuge tube in step (3), it is left that 5min is placed in 50 DEG C of water-baths The right side constantly mildly spins upside down centrifuge tube therebetween, to ensure that blob of viscose sufficiently dissolves；
(5) column equilibration: into adsorption column CB2,500 μ L equilibrium liquid BL, centrifugation is added in (adsorption column is placed in advance in collecting pipe) 12,000rpm/min, 1min outwell the waste liquid in collecting pipe, adsorption column are placed back in collecting pipe；
(6) step (5) acquired solution is added in adsorption column CB2, stands 2min, 10,000rpm/min, centrifugation 30s, Fall the waste liquid in collecting pipe, then adsorption column CB2 is put into collecting pipe；
(7) 600 μ L rinsing liquid PW buffer are added into adsorption column, stand 3min, are centrifuged 10,000rpm/min, 30s, The waste liquid in collecting pipe is outwelled, adsorption column CB2 is put into collecting pipe；
(8) step (7) are repeated；
(9) suction attached column is centrifuged, 12,000rpm/min, 2min, as far as possible removing rinsing liquid, and adsorption column is placed in room temperature and is put 10min is set, is thoroughly dried；
(10) adsorption column CB2 is put into collecting pipe, 50 μ L Elution is vacantly added dropwise to adsorbed film middle position Buffer (65 DEG C of preheatings), stands 3min, is centrifuged 12,000rpm/min, 2min；
(11) from centrifuge tube in step (10) is taken out in centrifuge, intermediate adsorption column CB2 is abandoned, centrifuge tube lid is covered Son retains the DNA sample in centrifuge tube；
(12) DNA sample in step 11 is placed in 4 DEG C of preservations, prepares agarose gel electrophoresis identification glue and recycles DNA piece Section.
2.2PCR product and carrier double enzyme digestion reaction
(1) label needs well the 1.5mL EP used to manage, and sample-adding is carried out according to the following table in 1.5mL EP pipe, mixes: 50 μ L reaction system
(2) the 1.5mL EP pipe in step (1) is placed in corresponding enzyme optimum temperature thermostat water bath, water-bath 2-3h.
The recycling of double enzyme digestion product glue: taking out above-mentioned double digestion system, carries out agarose gel electrophoresis to recycle DNA therein Segment, method are recycled with PCR product glue in 1.2.1.
2.3 connection reactions
(1) it is several to prepare clean 1.5mL EP pipe, marks, is placed on EP pipe support stand-by.
(2) sample-adding, mixing is carried out according to the following table in 1.5mL EP pipe.
(3) after completing sample-adding according to table in step (2), each 10 μ l reaction system is placed in 16 DEG C of cryogenic liquids and is followed In ring machine, water-bath 10-16h；
(4) EP pipe in step (3) is taken out, is placed it in 65 DEG C of water-baths, water-bath 15min；
(5) the EP pipe in step (4) is taken out, 4 DEG C of preservations are placed in.
2.4 conversion reaction
(1) 10 μ L connection reaction solutions are rapidly joined in 100 μ L competent cells, and blows and beats mixing, ice bath 30min；
(2) sample cell is taken out, is placed in 42 DEG C of water-bath 100s, immediately after ice bath 2min；
(3) it takes out sample cell and 600 μ L LB liquid mediums is added into sample cell, then by sample in superclean bench Quality control is placed in 37 DEG C of constant-temperature tables, and 220rpm/min cultivates 1h；
(4) coated plate: taking out sample cell in step (3), and room temperature is centrifuged 8,000rpm/min, 2min, removes 600 μ L supernatants The thallus of bottom of the tube is resuspended in body, remaining supernatant, and the bacterium solution of resuspension is put into corresponding conversion plate center, will be turned with bacteria stick is applied The bacterium solution for changing plate center is uniformly spread out.
(5) step of converting (4) plate is just being placed in biochemical constant incubator, after 37 DEG C of culture 1h, flat-plate inverted will be converted It sets and carries out culture 15h；
(6) conversion results are observed.
2.5 plasmid extractions and double digestion are identified
2.5.1 plasmid extraction
(1) with 10 μ L liquid transfer gun heads from conversion plate in picking monoclonal to the 5mL resistance of benzyl containing ammonia LB liquid medium In, 37 DEG C, 220rpm/min shakes bacterium and stays overnight；
(2) bacterium solution is moved in 1.5mL EP pipe, room temperature centrifugation, 12,000rpm/min, 2min abandon supernatant；
(3) 250 μ L plasmids are added into the EP pipe of step (2) and extract reagent P1buffer, thorough suspension thalline；
(4) 250 μ L P2buffer are added into step (3) solution, immediately mildly 5-10 mixing of reverse centrifuge tube, room Temperature stands 2-4min；
(5) 350 μ L P3buffer are added into step (4) solution, immediately mildly 5-10 mixing of reverse centrifuge tube；Room Temperature stands 2-4min；
(6) by step (5) solution, room temperature centrifugation, 14,000rpm/min, 10min；
(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s are outwelled Liquid in collecting pipe；
(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s outwell receipts Liquid in collector；
(9) 500 μ L wash solution are added to adsorption column center, room temperature is centrifuged, 12,000rpm/min, 30s, Fall liquid in collecting pipe, is repeated once；
(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.
(11) adsorption column is put into a clean 1.5mL centrifuge tube, 30 μ L Elution is added to absorption center membrane Buffer is stored at room temperature 5min, room temperature centrifugation, 12,000rpm, 2min.Save DNA solution in pipe.
2.5.2 double digestion is identified
(1) label needs well the 1.5mL EP used to manage, and sample-adding: 20 μ L reaction systems is carried out according to the following table
(2) the 20 μ L reaction system of EP pipe in step (1) is placed in 37 DEG C of thermostat water baths, water-bath 2h.
(3) the double digestion system sample in step (2) is subjected to agarose gel electrophoresis, whether checks Insert Fragment size Correctly；Experimental result is shown in Fig. 2: digestion identification building is correct.
(4) selection Insert Fragment, which is correctly cloned, send sequencing company to be sequenced.
2.6 endotoxin-free plasmids mention greatly
2.6.1 endotoxin-free plasmid is extracted
(1) correctly clone is seeded in the culture medium of the 100mL resistance of benzyl containing ammonia for sequencing, in 37 DEG C of constant-temperature tables, 220rpm/min cultivates 15h；
(2) bacterium solution cultivated in step (1) is transferred in 50mL centrifuge tube, room temperature 8,000rpm/min, centrifugation 5min, Thallus is collected, supernatant culture medium is discarded；
(3) 8mL solution P1 is added into the centrifuge tube of step (2), thallus is sufficiently resuspended with pipettor；
(4) 8mL solution P2 is added into the centrifuge tube of step (3), mildly overturns centrifuge tube 6-8 times, is stored at room temperature immediately 5min；
(5) 8mL solution P4 is added into the centrifuge tube of step (4), turns upside down 6-8 times, is mixed well to solution immediately There is white flock precipitate, is placed at room temperature for 10min or so.8,000rpm/min room temperatures are centrifuged 5-10min, make white precipitate from extremely Tube bottom；
(6) by supernatant in step (5) all careful immigration filter CS1, slowly push handle filter, filtrate collection exist In clean 50mL centrifuge tube；
(7) column equilibration: into adsorption column CP6, the equilibrium liquid BL of 2.5mL, room is added in (adsorption column is put into 50mL collecting pipe) 8,000rpm/min of temperature is centrifuged 2min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；
(8) isopropanol of 0.3 times of filtrate volume is added into step (6) filtrate, is transferred to absorption after mixing of turning upside down In column CP6.Room temperature 8,000rpm/min are centrifuged 2min, outwell liquid in collecting pipe, adsorption column CP6 is reentered into the same receipts In collector；
(9) 10mL rinsing liquid PW, room temperature 8 are added into step (8) adsorption column CP6,000rpm/min is centrifuged 2min, abandons and receives Waste liquid in collector, adsorption column is placed back in collecting pipe；
(10) repetitive operation step (9) is primary；
(11) 3mL dehydrated alcohol, room temperature 8 are added into step (10) adsorption column CP6,000rpm/min is centrifuged 2min, Fall waste liquid；
(12) step (11) adsorption column CP6 is placed back in collecting pipe, room temperature 8,000rpm/min is centrifuged 5min.It will inhale Attached column CP6 uncaps, and is placed in and is placed at room temperature for several minutes and dries；
(13) adsorption column in step (12) is put into clean 50mL centrifuge tube, it is slow that 1-2mL is added in adsorbed film center Fliud flushing TB is stored at room temperature 5min, room temperature 8, and 000rpm/min is centrifuged 2min, and the eluent in 50mL centrifuge tube is all moved into one A clean 1.5mL centrifuge tube surveys concentration, -20 DEG C of preservations.
(14) it takes the obtained plasmid DNA solution of 1-2 μ L to carry out agarose gel electrophoresis and saves electrophoresis result data.
The foundation of embodiment 3:pEE12.4-OPTI-gD Transfected Recombinant Plasmid CHO-K1 cell and monoclonal screening
3.1CHO-K1 cell transfecting
(1) prepare: Biohazard Safety Equipment ultraviolet sterilization 30min；DMEM/F12 (containing 10% serum, 1% is dual anti-), DMEM/F12 37 DEG C of water-baths, which are placed in, with PBS is preheated to 37 DEG C.
(2) cell (10cm Tissue Culture Dish) is taken out from 37 DEG C of incubators, culture medium is discarded supernatant, with the 8mL of pre-temperature It is primary that PBS washes cell, and discards PBS.
(3) 1-2mL 0.25%trypsin-EDTA is added in each 10cm Tissue Culture Dish, and room temperature digests 2min or so, shows Micro- microscopic observation cell shrinkage is rounded, and is in individual cells.
(4) 4mL DMEM/F12 (contain 10% serum, 1% is dual anti-) is added and terminates digestion reaction, and with pipettor by cell It dispels.
(5) cell digested is transferred in 15mL centrifuge tube, room temperature centrifugation, 200g, 5min.
(6) it with DMEM/F12 (containing 10% serum, 1% is dual anti-) again suspension cell, counts.
(7) diluting cells are to 2 × 105A/mL, the cell for taking 2mL to mix are added to six orifice plates, and six orifice plates are placed into 37 DEG C, 5%CO2It is incubated overnight in cell incubator.
(8) step (7) Tissue Culture Dish is taken out, observes cell state: when cell degree of crossing reaches 80%-90% Start to transfect, culture medium is changed into the DMEM/F12 of antibiotic-free serum-free, the hole 2mL/ before transfection.
(9) it dilutes plasmid: diluting plasmid with OPTI-MEM, 2.5 μ g plasmids are added in 125 μ L OPTI-MEM, are then added 2.5 μ L plus mix, are stored at room temperature 5min.
(10) it dilutes in Lipofectamine LTX:125 μ L OPTI-MEM and 9 μ L Lipofectamine LTX is added, Then 2.5 μ L plus are added, mixes gently, is stored at room temperature 5min.
(11) step (10) and step (11) mixture are mixed gently.It is placed at room temperature for 5min, six holes are then added dropwise It is uniformly distributed in plate.
(12) six orifice plates are placed in 37 DEG C, 5%CO24-6h is cultivated in cell incubator.
(13) it changes liquid: discarding supernatant culture medium, 2mL DMEM/F12 (dual anti-containing 10% serum 1%) is added, by six orifice plates 37 DEG C are placed in, 5%CO2It is cultivated in cell incubator.
3.2 pressurization screenings
Start to pressurize for 24 hours after transfection: taking out six orifice plate cells from 37 DEG C of incubators, discard supernatant culture medium, 2mL is added DMEM/F12 (containing 10% serum and 25 μM of MSX), pressurize 7d, and centre observation cell, dead cell changes liquid more.
The screening of 3.3 monoclonals
(1) when death ray basic to negative control cell is screened in pressurization, about 7days starts monoclonal screening.
(2) six orifice plates are taken out, culture medium is discarded, PBS is washed once, 300 μ L 0.25%trypsin-EDTA are then added, Room temperature digests 2min or so, and 2mL DMEM/F12 (containing 10% serum and 25 μM of MSX) is added and terminates digestion reaction, and uses liquid relief Device dispels cell.
(3) cell digested is transferred in 15mL centrifuge tube, room temperature centrifugation, 200g, 5min.
(4) it with DMEM/F12 (containing 10% serum and 25 μM of MSX) again suspension cell, counts.
(5) bed board: diluting cells to 5/mL, the cell for taking 200 μ L to mix are added in 96 orifice plates, are placed into 37 DEG C, 5%CO24-6h is incubated in cell incubator.
(6) hole of individual cells is recorded.
(7) when the hole length of individual cells in 96 orifice plates is got up, culture medium is discarded, PBS is washed once, and 100 μ are added L0.25%trypsin-EDTA, room temperature digest 2min or so, and 2mL DMEM/F12 (containing 10% serum and 25 μM of MSX) is added eventually Only digestion reaction, and dispelled cell with pipettor.Cell liquid is transferred to 12 orifice plates, when 12 orifice plates cover with, takes supernatant, Whether ELISA detection clone is the positive, and the positive colony of high efficient expression continues to expand culture, freeze.
The domestication of embodiment 4:CHO-K1 cell strain is cultivated at suspending
(1) prepare: Biohazard Safety Equipment ultraviolet sterilization 30min；DMEM/F12 (containing 10% serum, 25 μM of MSX) is placed in 37 DEG C 37 DEG C are preheated in water-bath.
(2) cell (10cm Tissue Culture Dish) is taken out from 37 DEG C of incubators, culture medium is discarded supernatant, with the 8mL of pre-temperature It is primary that PBS washes cell, and discards PBS.
(3) 1-2mL0.25%trypsin-EDTA is added in each 10cm Tissue Culture Dish, and room temperature digests 2min or so, shows Micro- microscopic observation cell shrinkage is rounded, and is in individual cells.
(4) 4mL DMEM/F12 (contain 10% serum, 25 μM of MSX) is added and terminates digestion reaction, and with liquid-transfering gun by cell It dispels.
(5) cell digested is transferred in 15mL centrifuge tube, room temperature centrifugation, 200g, 5min.
(6) it with 100%DMEM/F12 (containing 10% serum, 25 μM of MSX) suspension cell, counts.
(7) diluting cells are to 5 × 105A cell/mL inoculation 30mL culture is based in a 125mL shaking flask.Cell culture Bottle is placed into 37 DEG C, 5%CO2120rpm/min is incubated overnight on rail mounted oscillator in cell incubator.
(8) bio-safety counter top is sterilized with 75% alcohol wipe, ultraviolet irradiation 30min.
(9) every counting cell density and vigor for 24 hours.
(10) second generation culture is carried out when cell survival rate reaches 94-97% after first generation cell culture is primary.
(11) prepare: Biohazard Safety Equipment ultraviolet sterilization 30min；100%DMEM/F12 (contains 10% serum, 25 μM of MSX), EX-CELL 302 is placed in CO237 DEG C are preheated in cell incubator.
(12) it takes out cell from 37 DEG C of incubators to be transferred in 50mL centrifuge tube, room temperature 200g is centrifuged 5min.
(13) DMEM/F12 (containing 10% serum, 25 μM of MSX) and EX-CELL 302 are mixed by 1:1, is suspended again thin Born of the same parents count.
(14) diluting cells are to 5 × 105A cell/mL inoculation 30mL culture is based in a 125mL shaking flask.Cell culture Bottle is placed into 37 DEG C, 5%CO2120rpm/min is incubated overnight on rail mounted oscillator in cell incubator.
(15) bio-safety counter top is sterilized with 75% alcohol wipe, ultraviolet irradiation 30min.
(16) every counting cell density and vigor for 24 hours.
(17) cell survival rate that second generation culture obtains afterwards twice is greater than 95%；Third is commissioned to train to support to six and be obtained afterwards three times Cell survival rate be greater than 95%.After 7 weeks, cell inoculation breeds three generations after 3 days, and density reaches 1 × 106A cell/mL, simultaneously Cell survival rate reaches 95%, which is considered being already adapted to the culture that suspends.Inoculum density is reduced to 3 × 105A/mL.
(18) through taming, 3H8,5G3,7G11,7D2,4C2 and 6A3 monoclonal cell strain is all satisfied requirement, show domestication at Function.
Embodiment 5: cell shake flask fermentation
(1) optimize fermentation medium: using only the Ex-cell302 of the CD-CHO and 40% of Ex-cell302 or 60% Mixed culture medium carries out shake flask fermentation verifying to above-mentioned 6 kinds of cells.It is using only the cell number of Ex-cell302 fermentation 3H8,5G3,7G11,7D2,4C2,6A3；Utilize the list of the Ex-cell302 mixed culture medium fermentation of 60% CD-CHO and 40% Clonal cell line number is 3H8-C, 5G3-C, 7G11-C, 7D2-C, 4C2-C, 6A3-C.
(2) shaking flask cell is taken out from CO2 constant-temperature table, is counted.
(3) diluting cells are to 2.5-3.5 × 105A cell/mL inoculation 30mL culture is based in a 125mL shaking flask.Carefully Born of the same parents' culture bottle is placed into 37 DEG C, and 100rpm/min is incubated overnight in 5%CO2 constant-temperature table.
(4) every counting cell density and vigor for 24 hours, glucose is surveyed, when blood glucose is lower than 2g/L, adds glucose To 4g/L；1mL sample is taken daily, and supernatant is for detecting protein expression situation.
(5) feed supplement (the about the 4th day): supplement 79.6g/L CD Efficient Feed C AGT adds basal medium 10%.
Start within (6) the 5th days, by CO2Incubator temperature is adjusted to 32 DEG C.
(7) the 9th days, 79.6g/L CD Efficient Feed C AGT is supplemented, the 10% of basal medium is added.
(8) the 12nd days, harvest cell conditioned medium.
(9) SDS-PAGE detects the expression of monoclonal cell strain PRV-gD albumen.As shown in figure 3, swimming lane 1 is Marker, swimming lane 2 are the positive control albumen of 1 μ g, other are the monoclonal cell using the fermentation of above two different culture medium Strain culture supernatant.Wherein 6A3-C (using 60% CD-CHO and 40% 302 mixed culture medium of Ex-cell ferment, wherein 60% and 40% be volume ratio) porcine pseudorabies virus gD protein yield highest, preresearch estimates, expression yield be up to 2-3g/ L is suitble to needed for large-scale production.Wherein 302 mixed culture medium of Ex-cell of 60% CD-CHO and 40% is than being used alone 302 culture medium fermentation yield of Ex-cell wants high.
Embodiment 6: protein purification
(1) GE excle filler 4ml is taken, with BufferA (20mM NaH2PO4, 500mM NaCl, 0.05%Tween 20, PH 7.4) 5 column volumes of balance；
(2) 4ml filler is added in cell conditioned medium, in 4 DEG C of mixing 1h on roller bottle machine；
(3) filler and cell conditioned medium are transferred in sky chromatographic column, flow through cell conditioned medium；
(4) it washs: being eluted with the BufferA of the imidazoles containing 20mM, each 3ml, in mixing 15min on rotation vortex mixer, washed To Coomassie brilliant blue reagent without chromogenic reaction, 80 μ l is taken to keep sample detection.
(5) it elutes: being eluted with the BufferA of the imidazoles containing 250mM, each 3ml, in mixing 15min on rotation vortex mixer, washed To Coomassie brilliant blue reagent without chromogenic reaction, 80 μ l is taken to keep sample detection.
(6) it washs: being eluted with the BufferA of the imidazoles containing 500mM, each 3ml, in mixing 15min on rotation vortex mixer, washed To Coomassie brilliant blue reagent without chromogenic reaction, 80 μ l is taken to keep sample detection.
(7) dialysis change liquid: the imidazole elution containing destination protein is poured into bag filter, with 1 × PBS dialysis at least 1, 000 times, 80 μ l is taken to keep sample detection.
(8) Fig. 4 show 6A3 monoclonal cell strain supernatant porcine pseudorabies virus gD protein purification testing result.
(9) protein concentration, 6A3,4C2 and 7D2 monoclonal cell strain protein concentration and purity testing: are measured using BCA method Albumen yield is about 1.5-3g/L；Purity is detected using HPLC method, purity is attained by 90% or more.
The preparation of 7 pig GM-CSF albumen of embodiment
The preparation of pig GM-CSF albumen referring in particular to the applicant apply application No. is 201710343196.8 inventions The preparation method of patent application document.
Embodiment 8: vaccine preparation and immunization experiment
8.1 vaccine preparation
The porcine pseudorabies virus gD albumen of appropriate CHO-K1 cell expression and pig GM-CSF albumen are added to ISA201VG (antigen phase and adjuvant weight ratio are 1:1, wherein antigen is mutually the porcine pseudorabies virus gD egg of CHO-K1 cell expression in adjuvant White and pig GM-CSF albumen), emulsification, quality inspection qualification are placed on 4 DEG C of preservations；Specific vaccine information is as shown in the table:
8.2 immunization experiment
4-5 week old piggy 65 (PRV antigen-antibody is negative) is screened, is randomly divided into 10 groups, every group 5, one group is control Group, control group pig muscle injection 1ml PBS；One group is used as market seedling immunized controls group, is immunized market seedling (marker vaccines), One exempt from 3 weeks after booster immunization it is primary；Remaining 11 groups are used as subunit vaccine immune group, and immune group pig successively distinguishes intramuscular injection The vaccine 1- vaccine 11 of 8.1 preparations, is immunized 2 times, and the 3 weeks booster immunizations in interval are primary.Respectively before immune, two exempt from before and two exempt from after 14 days acquisition serum detects antibody titer.As a result as shown in Figure 5: 1) from the point of view of the variation of the potency of vaccine 1- vaccine 4, with gD The increase of protein concentration, potency increased, but increased amplitude is little；2) become from the potency of vaccine 2, vaccine 5- vaccine 8 From the point of view of changing, after the vaccine (vaccine 5- vaccine 8) containing pig GM-CSF albumen is either exempted from one or after two exempt from, the height of potency Potency after spending obvious height than vaccine (vaccine 2) without containing pig GM-CSF albumen and being attained by or be immunized higher than market seedling Level, but with the increase of pig GM-CSF protein concentration, the increased amplitude of potency is not obvious；3) from vaccine 1 and vaccine 9, Or from the point of view of the potency variation of vaccine 3 and vaccine 10 or vaccine 4 and vaccine 11, vaccine (vaccine 9, epidemic disease containing pig GM-CSF albumen Seedling 10, vaccine 11) potency obviously than without pig GM-CSF albumen vaccine (vaccine 1, vaccine 3, vaccine 4) potency than It is high；3) good antibody titer on the whole, can be generated after 12 groups of vaccine immunities, and potency is attained by after exempting from two 28000 or more, 11 groups of subunit vaccines exempt from two after antibody titer level and market seedling it is equal or higher, in addition, containing Potency of the potency of the vaccine of pig GM-CSF albumen obviously than the vaccine without pig GM-CSF albumen is high, this illustrates that pig is added The immune effect of the subunit vaccine can be significantly improved after GM-CSF albumen.
8.3ELISA detects antibody titer
(1) it is coated with: the gD albumen for using coating buffer (50mM carbonate buffer solution, pH 9.5) dilution to purify to 2 μ g/ml, 100 μ l are added in every hole on ELISA Plate, and 4 DEG C of refrigerators are stood overnight after sealed membrane is sealed；
(2) it washs: after refrigerator taking-up ELISA Plate, being put into board-washing machine and wash, cleaning solution PBST；
(3) close: 200 μ l confining liquids (5% defatted milk), 37 DEG C of incubation 2h after sealed membrane is sealed are added in every hole；
(4) preparation of samples: by known information and needing dosage, and serum is carried out appropriate dilution with confining liquid；
(5) it washs: with (2)；
(6) it is loaded: dilute serum is added, while being negative control, 37 DEG C of incubation 1h with confining liquid；
(7) it washs: with (2)；
(8) add secondary antibody: secondary antibody 100 the μ l, 37 DEG C of incubation 0.5h of the diluted HRP label of appropriateness is added in every hole；
(9) it washs: with (2)；
(10) develop the color: the TMB developing solution of 100 μ l, 37 DEG C of incubation 10min are added in every hole under the conditions of being protected from light；
(11) terminate: 50 μ l terminate liquid (2M H are added in every hole2SO4), terminate reaction；
(12) it detects: measuring sample OD value in 450nm wavelength, analyze data；
(13) interpretation of result: judge the standard of antibody positive: P/N >=2.1, OD450 >=0.1.
The present invention is illustrated by above embodiment, it is understood, however, that the present invention is not limited to institutes here The particular example and embodiment of description.Purpose herein comprising these particular examples and embodiment is to help this field In technical staff practice the present invention.Any those of skill in the art are easy to do not departing from spirit and scope of the invention In the case of be further improved and perfect, therefore the present invention is only by the content of the claims in the present invention and the limit of range System, intention, which covers, all to be included the alternative in the spirit and scope of the invention as defined by appendix claim and waits Same scheme.
Sequence table
<110>Zhejiang oceanic rise Biotechnology Co., Ltd
<120>preparation method of porcine pseudorabies virus gD albumen and porcine pseudorabies virus subunit vaccine and application
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 1020
<212> DNA
<213>the PRV gD gene order after codon optimization
<400> 1
atggctgacg tggatgctgt gcctgctcca accttcccac ctccagctta cccatatacc 60
gagagctggc agctgacact gaccacagtg ccctctcctt ttgtgggacc agctgacgtg 120
taccacacaa ggccactgga ggatccatgc ggagtggtgg ctctgatctc tgacccccag 180
gtggatagac tgctgaacga ggctgtggct cacaggcggc ctacctacag ggctcatgtg 240
gcctggtatc ggatcgctga cggctgtgcc catctgctgt acttcatcga gtatgctgac 300
tgcgatccta gacagatctt tggccgctgt agacgcagga ccacacctat gtggtggacc 360
ccatccgccg actacatgtt ccctacagag gatgagctgg gcctgctgat ggtggctcca 420
ggcaggttta acgagggcca gtatcggaga ctggtgagcg tggacggcgt gaatatcctg 480
accgatttca tggtggccct gccagaggga caggagtgcc cttttgctag ggtggaccag 540
caccggacat acaagttcgg cgcctgttgg tccgacgata gctttaagag aggcgtggat 600
gtgatgcgct tcctgacccc tttttatcag cagccccctc atcgggaggt ggtgaactac 660
tggtatcgca agaatggaag gaccctgcca agggcttacg ctgctgctac accctatgct 720
atcgacccag ctagaccttc tgctggatcc ccaaggccaa ggcctaggcc aagaccaagg 780
cctaggccaa agccagagcc tgctccagct accccagctc caccaggcag actgccagag 840
cctgctacaa gggatcacgc tgctggaggc aggccaaccc caaggcctcc aagaccagag 900
acaccacata ggcctttcgc tccacctgct gtggtgcctt ccggatggcc tcagccagcc 960
gagccatttc cacccaggac cacagctgcc cccggcgtgc atcaccatca ccatcactaa 1020
<210> 2
<211> 1020
<212> DNA
<213>the PRV gD gene order before codon optimization
<400> 1
atggcggacg tggacgccgt gcccgcgccg accttccccc cgcccgcgta cccgtacacc 60
gagtcgtggc agctgacgct gacgacggtc ccctcgccct tcgtcggccc cgcggacgtc 120
taccacacgc gcccgctgga ggacccgtgc ggggtggtgg cgctgatctc cgacccgcag 180
gtggaccggc tgctgaacga ggcggtggcc caccggcggc ccacgtaccg cgcccacgtg 240
gcctggtacc gcatcgcgga cgggtgtgcg cacctgctgt actttatcga gtacgccgac 300
tgcgacccca ggcagatctt tgggcgctgc cggcgccgca ccacgccgat gtggtggacc 360
ccgtccgcgg actacatgtt ccccacggag gacgagctgg ggctgctcat ggtggccccg 420
gggcggttca acgagggcca gtaccggcgc ctggtgtccg tcgacggcgt gaacatcctc 480
accgacttca tggtggcgct ccccgagggg caagagtgcc cgttcgcccg cgtggaccag 540
caccgcacgt acaagttcgg cgcgtgctgg agcgacgaca gcttcaagcg gggcgtggac 600
gtgatgcgat tcctgacgcc gttctaccag cagcccccgc accgggaggt ggtgaactac 660
tggtaccgca agaacggccg gacgctcccg cgggcctacg ccgccgccac gccgtacgcc 720
atcgaccccg cgcggccctc ggcgggctcg ccgaggccca ggccccggcc ccggcccagg 780
ccccggccga agcccgagcc cgccccggcg acgcccgcgc cccccggccg cctgcccgag 840
ccggcgacgc gggaccacgc cgccgggggg cgccccacgc cgcgaccccc gaggcccgag 900
acgccgcacc gccccttcgc cccgccggcc gtcgtgccca gcgggtggcc gcagcccgcg 960
gagccgttcc cgccccggac caccgccgcg ccgggcgtcc atcaccatca ccatcactaa 1020
<210> 3
<211> 339
<212> PRT
<213>PRV gD protein sequence
<400> 1
Met Ala Asp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala
1 5 10 15
Tyr Pro Tyr Thr Glu Ser Trp Gln Leu Thr Leu Thr Thr Val Pro Ser
20 25 30
Pro Phe Val Gly Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu Asp
35 40 45
Pro Cys Gly Val Val Ala Leu Ile Ser Asp Pro Gln Val Asp Arg Leu
50 55 60
Leu Asn Glu Ala Val Ala His Arg Arg Pro Thr Tyr Arg Ala His Val
65 70 75 80
Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu Leu Tyr Phe Ile
85 90 95
Glu Tyr Ala Asp Cys Asp Pro Arg Gln Ile Phe Gly Arg Cys Arg Arg
100 105 110
Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro
115 120 125
Thr Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn
130 135 140
Glu Gly Gln Tyr Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu
145 150 155 160
Thr Asp Phe Met Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala
165 170 175
Arg Val Asp Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp
180 185 190
Asp Ser Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro Phe
195 200 205
Tyr Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg Lys
210 215 220
Asn Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala Thr Pro Tyr Ala
225 230 235 240
Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg Pro Arg
245 250 255
Pro Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala Pro Ala Thr Pro
260 265 270
Ala Pro Pro Gly Arg Leu Pro Glu Pro Ala Thr Arg Asp His Ala Ala
275 280 285
Gly Gly Arg Pro Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg
290 295 300
Pro Phe Ala Pro Pro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala Glu
305 310 315 320
Pro Phe Pro Pro Arg Thr Thr Ala Ala Pro Gly Val His His His His His
325 330 335
His

Claims (10)

1. a kind of preparation method of porcine pseudorabies virus gD albumen, which is characterized in that the preparation method comprises the following steps:
1) it by the porcine pseudorabies virus gD protein gene cloning after codon optimization into carrier for expression of eukaryon, obtains containing pig puppet The recombinant plasmid of rabies viruses gD protein coding gene；
2) again by the Transfected Recombinant Plasmid into Chinese hamster ovary celI, Chinese hamster ovary celI strain is obtained；
3) by Chinese hamster ovary celI strain described in culture, screening, domestication step 2), the cell strain that height is expressed is obtained；
4) cell strain highly expressed described in fermented and cultured step 3) obtains recombinant porcine pseudorabies poison gD albumen after purification.
2. preparation method according to claim 1, which is characterized in that the porcine pseudorabies virus gD after the codon optimization Protein coding gene is as shown in SEQ ID NO.1.
3. preparation method according to claim 1, which is characterized in that the carrier for expression of eukaryon is pEE12.4.
4. preparation method according to claim 1, which is characterized in that the Chinese hamster ovary celI is CHO-K1 cell.
5. preparation method according to claim 1, which is characterized in that in the step 4), institute in fermented and cultured step 3) The culture medium used when stating the cell strain of height expression is the mixed culture medium of CD-CHO and Ex-cell 302, the mixing training Supporting the volume ratio of CD-CHO and Ex-cell 302 in base is 6:4.
6. a kind of porcine pseudorabies virus subunit vaccine, which is characterized in that the porcine pseudorabies virus subunit vaccine includes:
(1) containing the porcine pseudorabies virus gD albumen by any method preparation of Claims 1 to 5, the pseudorabies The concentration of viral gD albumen is g/ parts of 30~200 μ；
(2) pig GM-CSF albumen, the concentration of the pig GM-CSF albumen are g/ parts of 10~80 μ；And
(3) pharmaceutically acceptable adjuvant.
7. porcine pseudorabies virus subunit vaccine according to claim 6, which is characterized in that the porcine pseudorabies virus gD The concentration of albumen is g/ parts of 50 μ~g/ parts of 100 μ.
8. porcine pseudorabies virus subunit vaccine according to claim 6, which is characterized in that the pig GM-CSF albumen is dense Degree is g/ parts of 20 μ~g/ parts of 40 μ.
9. porcine pseudorabies virus subunit vaccine according to claim 6, which is characterized in that described pharmaceutically acceptable Adjuvant is ISA 201VG, and the ISA 201VG and the weight ratio of antigen phase are 1:1, and the antigen is mutually the porcine pseudorabies Malicious gD albumen and the pig GM-CSF albumen.
10. a kind of any according to claim 1~9 the porcine pseudorabies virus gD albumen and porcine pseudorabies virus subunit epidemic disease Application of the seedling in preparation and dependent diagnostic reagent.
CN201710551363.8A 2017-07-07 2017-07-07 Preparation method of porcine pseudorabies virus gD protein, porcine pseudorabies virus subunit vaccine and application Pending CN109206491A (en)

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CN109943592A (en) * 2019-04-29 2019-06-28 华中农业大学 Recombinant baculovirus transfer vector, recombinant baculovirus and the preparation method and application of the protein gene of gD containing porcine pseudorabies virus
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CN110156878A (en) * 2019-05-24 2019-08-23 北京标驰泽惠生物科技有限公司 Porcine pseudorabies virus gE-gI albumen and its expression plasmid, preparation method and application
CN112142827A (en) * 2019-06-28 2020-12-29 浙江海隆生物科技有限公司 gB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application thereof
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CN110227153A (en) * 2019-07-17 2019-09-13 苏州世诺生物技术有限公司 A kind of preparation method and applications of porcine rotavirus subunit vaccine
CN114574503A (en) * 2022-03-14 2022-06-03 成都史纪生物制药有限公司 Hog cholera E2 protein gene, porcine pseudorabies virus gD protein gene and application thereof
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CN114908056A (en) * 2022-05-18 2022-08-16 华中农业大学 Recombinant CHO cell line for expressing porcine PRV gDFc or gBFc fusion protein and construction method and application thereof
CN114908056B (en) * 2022-05-18 2024-04-16 华中农业大学 Recombinant CHO cell line expressing pig PRV gDFc or gBFc fusion protein and construction method and application thereof

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